Compositions and methods for detection of mycobacterium tuberculosis

ABSTRACT

Method for the rapid detection of the presence or absence of  Mycobacterium tuberculosis  (MTB) and other members of the MTB-complex in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers, probes targeting the esxJ gene, along with kits are provided that are designed for the detection of MTB.

FIELD OF THE INVENTION

The present disclosure relates to the field of bacterial diagnostics,and more particularly to detection of Mycobacterium Tuberculosis.

BACKGROUND OF THE INVENTION

Tuberculosis (TB) is a bacterial disease caused by various strains ofmycobacteria, such as Mycobacterium tuberculosis (MTB) and other membersof the MTB complex, most often found in the lungs. It is transmittedfrom person to person through the air when individuals with pulmonary orlaryngeal tuberculosis, cough, sneeze, or spit, and propel MTB into theair. It is estimated that one third of the world population is infectedwith MTB and 9 million people develop TB each year. TB continues to be aleading cause of human infectious disease and drug-resistant strains ofMTB are on the rise, especially in developing countries.

Two common first-line drugs for the treatment of MTB include isoniazid(INH) and rifampicin (RIF), and patients can acquire drug resistant MTBfrom living in or visiting a place where drug resistance is prevalent.Patients can also develop drug resistant MTB when their antibiotictreatment regimen is interrupted. Culturing in solid or liquid media isstill considered the gold standard for MTB and MTB drug resistancedetection, but culturing can take up to eight weeks for results. Thusthere is a need in the art for a quick and reliable method tospecifically detect MTB in a sensitive manner.

SUMMARY OR THE INVENTION

Certain embodiments in the present disclosure relate to methods for therapid detection of the presence or absence of MTB in a biological or nonbiological sample, for example, multiplex detection of MTB by real-timepolymerase chain reaction in a single test tube. Embodiments includemethods of detection of MTB comprising performing at least one cyclingstep, which may include an amplifying step and a hybridizing step.Furthermore, embodiments include primers, probes, and kits that aredesigned for the detection of MTB in a single tube. The detectionmethods are designed to target the esxJ gene (and its homologues genesesxK, esxM, esxP, esxW) which allows one to detect MTB in a single test.

In one embodiment, a method for detecting MTB in a sample is provided,including performing an amplifying step including contacting the samplewith a set of esxJ primers to produce an amplification product if MTB ispresent in the sample; performing a hybridizing step includingcontacting the amplification product with one or more detectable esxJprobes; and detecting the presence or absence of the amplified product,wherein the presence of the amplified product is indicative of thepresence of MTB in the sample and wherein the absence of the amplifiedproduct is indicative of the absence of MTB in the sample; wherein theset of esxJ primer comprises or consists of a sequence selected from thegroup consisting of SEQ ID NOs; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, and 21, or a complement thereof; andwherein the detectable esxJ probe comprises or consists of a sequenceselected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, or acomplement thereof.

In one embodiment, the primer set for amplification of the esxJ genetarget include a first primer comprising a first oligonucleotidesequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4,5, 6, 7, 8, and 9, or a complement thereof, and a second primercomprising a second oligonucleotide sequence selected from the groupconsisting of SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,and 21, or a complement thereof, and the detectable probe for detectionof the esxJ amplification product includes the nucleic acid sequences ofSEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, and 40, or a complement thereof.

Other embodiments provide an oligonucleotide comprising or consisting ofa sequence of nucleotides selected from SEQ ID NOs: 1-40, or acomplement thereof, which oligonucleotide has 100 or fewer nucleotides.In another embodiment, the present disclosure provides anoligonucleotide that includes a nucleic acid having at least 70%sequence identity (e.g., at least 75%, 80%, 85%, 90% or 95%, etc.) toone of SEQ ID NOs: 1-40, or a complement thereof, which oligonucleotidehas 100 or fewer nucleotides. Generally, these oligonucleotides may beprimer nucleic acids, probe nucleic acids, or the like in theseembodiments. In certain of these embodiments, the oligonucleotides have40 or fewer nucleotides (e.g. 35 or fewer nucleotides, 30 or fewernucleotides, etc.) In some embodiments, the oligonucleotides comprise atleast one modified nucleotide, e.g., to alter nucleic acid hybridizationstability relative to unmodified nucleotides. Optionally, theoligonucleotides comprise at least one label and/or at least onequencher moiety. In some embodiments, the oligonucleotides include atleast one conservatively modified variation. “Conservatively modifiedvariations” or, simply, “conservative variations” of a particularnucleic acid sequence refers to those nucleic acids, which encodeidentical or essentially identical amino acid sequences, or, where thenucleic acid does not encode an amino acid sequence, to essentiallyidentical sequences. One of skill will recognize that individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (typically lessthan 5%, more typically less than 4%, 2% or 1%) in an encoded sequenceare “conservatively modified variations” where the alterations result inthe deletion of an amino acid, addition of an amino acid, orsubstitution of an amino acid with a chemically similar amino acid.

In one aspect, amplification can employ a polymerase enzyme having 5′ to3′ nuclease activity. Thus, the donor fluorescent moiety and theacceptor moiety, e.g., a quencher, may be within no more than 5 to 20nucleotides (e.g., 8 or 10) of each other along the length of the probe.In another aspect, the esxJ probe includes a nucleic acid sequence thatpermits secondary structure formation. Such secondary structureformation generally results in spatial proximity between the first andsecond fluorescent moiety. According to this method, the secondfluorescent moiety on the probe can be a quencher.

The present disclosure provides for methods of detecting the presence orabsence of MTB in a biological sample from an individual. Such methodsgenerally include performing at least one cycling step, which includesan amplifying step and a dye-binding step. Typically, the amplifyingstep includes contacting the sample with a plurality of pairs of esxJprimers to produce one or more esxJ amplification products if an esxJnucleic acid molecule is present in the sample, and the dye-binding stepincludes contacting the esxJ amplification product with adouble-stranded DNA binding dye. Such methods also include detecting thepresence or absence of binding of the double-stranded DNA binding dyeinto the amplification product, wherein the presence of binding isindicative of the presence of MTB in the sample, and wherein the absenceof binding is indicative of the absence of MTB in the sample. Arepresentative double stranded DNA binding dye is ethidium bromide. Inaddition, such methods also can include determining the meltingtemperature between the esxJ amplification product and thedouble-stranded DNA binding dye, wherein the melting temperatureconfirms the presence or absence of MTB.

In a further embodiment, a kit for detecting one or more nucleic acidsof MTB is provided. The kit can include one or more sets of esxJ primersspecific for amplification of the esxJ gene target; and one or moredetectable esxJ probes specific for detection of the esxJ amplificationproducts.

In one aspect, the kit can include probes already labeled with donor andcorresponding acceptor moiety, e.g., another fluorescent moiety or adark quencher, or can include fluorophoric moieties for labeling theprobes. The kit can also include nucleoside triphosphates, nucleic acidpolymerase, and buffers necessary for the function of the nucleic acidpolymerase. The kit can also include a package insert and instructionsfor using the primers, probes, and fluorophoric moieties to detect thepresence or absence of MTB in a sample.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present subject matter, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows PCR growth curves of experiments using several forwardprimers specific for esxJ target for MTB.

FIG. 2 shows PCR growth curves of experiments using reverse primersspecific for esxJ target for MTB showing earlier elbow values and higherfluorescence from all three esxJ reverse primers compared to 16Soptimized oligos.

FIG. 3 shows PCR growth curves of experiments using several primersspecific for esxJ target for MTB, showing fluorescence and elbow valuesfrom 4 candidates with MTB target.

FIG. 4 shows PCR growth curves of experiments using several primersspecific for esxJ target for MTB, showing fluorescence from 4 candidateswith non-MTB target (M. gastri).

FIG. 5 shows PCR growth curves of experiments using several primersspecific for esxJ target for MTB, showing exclusivity demonstration withdilution series of MTB and 1e6c/PCR each of non-MTB species (M. gastri,M. szulgai, and M. kansasii).

DETAILED DESCRIPTION OF THE INVENTION

Diagnosis of MTB infection by nucleic acid amplification provides amethod for rapidly and accurately detecting the bacterial infection. Areal-time assay for detecting MTB in a sample is described herein.Primers and probes for detecting MTB are provided, as are articles ofmanufacture or kits containing such printers and probes. The increasedsensitivity of real-time PCR for detection of MTB compared to othermethods, as well as the improved features of real-time PCR includingsample containment and real-time detection of the amplified product,make feasible the implementation of this technology for routinediagnosis of MTB infections in the clinical laboratory.

The present disclosure includes oligonucleotide primers and fluorescentlabeled hydrolysis probes that hybridize to the ESX-5 gene locus of theMTB genome in order to specifically identify MTB using TaqMan®amplification and detection technology. The oligonucleotidesspecifically hybridize to the esxJ gene, located within the ESX-5 locus,and additionally hybridize to four other gene homologs (esxM, esxK,esxP, and esxW) located outside the ESX-5 locus elsewhere in the MTBgenome. Some members of the MTB-complex have less than five copies ofthis gene homologue. Having oligonucleotides that hybridize to multiplelocations in the genome is advantageous for improved sensitivitycompared to targeting a single copy genetic locus.

The multi-copy nature of many of the ESX-5 genes in the MTB genome hasbeen described in the literature (e.g., Uplekar et al., Infect. Immun.,(2001) 79(10):4042-4049). The proteins encoded by members of the ESX-5gene locus have been well studied and are believed to elicit a stronghost immune response that may be potentially useful for a future vaccineguarding against MTB infection. Oligonucleotides targeting esxJ and itshomologs for diagnostic purposes, however, have not been describedpreviously and can offer about 5-fold improved sensitivity compared witholigonucleotides targeting 16S (a single-copy MTB genetic locus).

The disclosed methods may include performing at least one cycling stepthat includes amplifying one or more portions of the esxJ nucleic acidmolecule gene target from a sample using one or more pairs of esxJprimers. “EsxJ primer(s)” as used herein refer to oligonucleotideprimers that specifically anneal to nucleic acid sequence encoding esxJ,and additionally hybridize to four other gene homologs (esxM, esxK,esxP, and esxW) in MTB, and initiate DNA synthesis therefrom underappropriate conditions producing the respective amplification products.Each of the discussed esxJ primers anneals to a target within oradjacent to the respective esxJ esxM, esxK, esxP, and esxW targetnucleic acid molecule such that at least a portion of each amplificationproduct contains nucleic acid sequence corresponding to the target. Theone or more of esxJ, esxM, esxK, esxP, and/or esxW amplificationproducts are produced provided that one or more of esxJ, esxM, esxK,esxP, and/or esxW nucleic acid is present in the sample, thus thepresence of the one or more of esxJ, esxM, esxK, esxP, and/or esxWamplification products is indicative of the presence of MTB in thesample. The amplification product should contain the nucleic acidsequences that are complementary to one or more detectable probes foresxJ. “esxJ probe(s)” as used herein refer to oligonucleotide probesthat specifically anneal to nucleic acid sequence encoding esxJ, andadditionally hybridize to four other gene homologs (esxM, esxK, esxP,and esxW) in MTB. Each cycling step includes an amplification step, ahybridization step, and a detection step, in which the sample iscontacted with the one or more detectable esxJ probes for detection ofthe presence or absence of MTB in the sample.

As used herein, the term “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid molecule (e.g., esxJ).Amplifying a nucleic acid molecule typically includes denaturing thetemplate nucleic acid, annealing primers to the template nucleic acid ata temperature that is below the melting temperatures of the primers, andenzymatically elongating from the primers to generate an amplificationproduct. Amplification typically requires the presence ofdeoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g.,Platinum® Taq) and an appropriate buffer and/or co-factors for optimalactivity of the polymerase enzyme (e.g., MgCl₂ and/or KCl).

The term “primer” as used herein is known to those skilled in the artand refers to oligomeric compounds, primarily to oligonucleotides butalso to modified oligonucleotides that are able to “prime” DNA synthesisby a template-dependent DNA polymerase, i.e., the 3′-end of the, e.g.,oligonucleotide provides a free 3′-OH group whereto further“nucleotides” may be attached by a template-dependent DNA polymeraseestablishing 3′ to 5′ phosphodiester linkage whereby deoxynucleosidetriphosphates are used and whereby pyrophosphate is released. Therefore,there is—except possibly for the intended function—no fundamentaldifference between a “primer”, an “oligonucleotide”, or a “probe”.

The term “hybridizing” refers to the annealing of one or more probes toan amplification product. Hybridization conditions typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

The term “5′ to 3′ nuclease activity” refers to an activity of a nucleicacid polymerase, typically associated with the nucleic acid strandsynthesis, whereby nucleotides are removed from the 5′ end of nucleicacid strand.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacreus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished.

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

The term “extension” or “elongation” when used with respect to nucleicacids refers to when additional nucleotides (or other analogousmolecules) are incorporated into the nucleic acids. For example, anucleic acid is optionally extended by a nucleotide incorporatingbiocatalyst, such as a polymerase that typically adds nucleotides at the3′ terminal end of a nucleic acid.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same, when compared and aligned for maximumcorrespondence, e.g., as measured using one of the sequence comparisonalgorithms available to persons of skill or by visual inspection.Exemplary algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST programs, which aredescribed in, e.g., Altschul et al. (1990) “Basic local alignment searchtool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification ofprotein coding regions by database similarity search” Nature Genet.3:266-272, Madden et al. (1996) “Applications of network BLAST server”Meth. Enzymol. 266:131-141, Altschul et al. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs” NucleicAcids Res. 25:3389-3402, and Zhang at al. (1997) “PowerBLAST: A newnetwork BLAST application for interactive or automated sequence analysisand annotation” Genome Res. 7:649-656, which are each incorporatedherein by reference.

A “modified nucleotide” in the context of an oligonucleotide refers toan alteration in which at least one nucleotide of the oligonucleotidesequence is replaced by a different nucleotide that provides a desiredproperty to the oligonucleotide. Exemplary modified nucleotides that canbe substituted in the oligonucleotides described herein include, e.g., aC5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7-propynyl-dA,a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, aC7-propargylamino-dA, a C7-propargylamino-AG, a7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, anitro pyrrole, a nitro indole, 2′-0-methyl Ribo-U, 2′-0-Ribo-C, anN4-ethyl-dC, an N6-methyl-dA, and the like. Many other modifiednucleotides that can be substituted in the oligonucleotides are referredto herein or are otherwise known in the art. In certain embodiments,modified nucleotide substitutions modify melting temperatures (Tm) ofthe oligonucleotides relative to the melting temperatures ofcorresponding unmodified oligonucleotides. To further illustrate,certain modified nucleotide substitutions can reduce non-specificnucleic acid amplification (e.g., minimize primer dimer formation or thelike), increase the yield of an intended target amplicon, and/or thelike in some embodiments. Examples of these types of nucleic acidmodifications are described in, e.g., U.S. Pat. No. 6,001,611, which isincorporated herein by reference.

Detection of MTB

The present disclosure provides methods to detect MTB by amplifying, forexample, a portion of the esxJ nucleic acid sequence. Nucleic acidsequences of esxJ is available (e.g., GenBank Accession No. NC_009565).Specifically, primers and probes to amplify and detect esxJ nucleic acidmolecule targets are provided by the embodiments in the presentdisclosure.

For detection of MTB, primers and probes to amplify the esxJ areprovided. EsxJ nucleic acids other than those exemplified herein canalso be used to detect MTB in a sample. For example, functional variantscan be evaluated for specificity and/or sensitivity by those of skill inthe art using routine methods. Representative functional variants caninclude, e.g., one or more deletions, insertions, and/or substitutionsin the esxJ nucleic acids disclosed herein.

More specifically, embodiments of the oligonucleotides each include anucleic acid with a sequence selected from SEQ ID NOs: 1-40, asubstantially identical variant thereof in which the variant has atleast, e.g., 80%, 90%, or 95% sequence identity to one of SEQ ID NOs:1-40, or a complement of SEQ ID NOs: 1-40 and the variant.

TABLE I EsxJ Forward Primers Forward Primers SEQ ID Oligo Name NO:Sequence Modifications JJESXFP01BBA 1 GTTTTGAGGTGCACGCCCAGJ J =t-butylbenzyldA JJESXFP02BBA 2 ATGGCCTCACGTTTTATGACGGJ J =t-butylbenzyldA JJESXFP03BBA 3 ACATGGCGGGCCGTTTTGJ J = t-butylbenzyldAJJESXFP04BBA 4 TTTTGAGGTGCACGCCCAGJ J = t-butylbenzyldA JJESXFP05BBA 5TTTGAGGTGCACGCCCAGJ J = t-butylbenzyldA JJESXFP06BBA 6TTGAGGTGCACGCCCAGJ J = t-butylbenzyldA JJESXFP07BBC 7GACGGTGGAGGACGAGGCTJ J = t-butylbenzyldC JJESXFP08BBC 8CGGTGGAGGACGAGGCTJ J = t-butylbenzyldC JJESXFP09BBC 9 GGTGGAGGACGAGGCTJJ = t-butylbenzyldC

TABLE II EsxJ Reverse Primers Reverse Primers SEQ ID Oligo Name NO:Sequence Modifications JJESXRP01BBA 10 ATGTTGCGAAACGCCTGATTCJ J =t-butylbenzyldA JJESXRP02BBA 11 TCGTAGTTGTTGGCGTCGCGAJ J =t-butylbenzyldA JJESXRP03BBA 12 TAGTTGTTGGCGTCGCGAACCJ J =t-butylbenzyldA JJESXRP04BBC 13 GGCCATGGTGTCTAGCGAGGTJ J =t-butylbenzyldC JJESXRP05BBC 14 GCCATGGTGTCTAGCGAGGTJ J =t-butylbenzyldC JJESXRP06BBC 15 CCATGGTGTCTAGCGAGGTJ J = t-butylbenzyldCJJESXRP07BBC 16 TCATGGTGTCTAGCGAGGTJ J = t-butylbenzyldC JJESXRP08BBC 17GTCATGGTGTCTAGCAGGTJ J = t-butylbenzyldC JJESXRP09BBC 18TGTCTAGCGAGGTCGCCTJ J = t-butylbenzyldC JJESXRP10BBC 19GTCTAGCGAGGTCGCCTJ J = t-butylbenzyldC JJESXRP11BBC 20 CCTCGGCCATGCCACTJJ = t-butylbenzyldC

TABBLE III EsxJ Probes Probes Oligo Name NO: Sequence ModificationsJJESXAP01FQ6 22 FTCTAGCQGAGGTCGCCTCGGCCAT P = phosphate, GCCACTCP F =th-FAM, Q = BHQ2 JJESXAP02FQ6 23 FTTTTGCQGCGGACGCCCACATCCG P =phosphate, GCP F = th-FAM, Q = BHQ2 JJESXSP01FQ6 24FGGCCGAQGGCGACCTCGCTAGAC P = phosphate, ACCATGACCTAGP F = th-FAM, Q =BHQ2 JJESXSP02FQ6 25 FTGGCCGQAGGCGACCTCGCTAGA P = phosphate,CACCATGACCTAGATP F = th-FAM, Q = BHQ2 JJESXAP02GFQ6 26FTTTTGCQGLGGACGCCCACATCCG P = phosphate, GCP F = th-FAM, Q = BHQ2,JJESXAP03FQ6 27 FTTTTGCQGCGGACGCCCACATCCG P = phosphate, GP F = th-FAM,Q = BHQ2 JJESXAP03GFQ6 28 FTTTTGCQGLGGACGCCCACATCCG P = phosphate, GPF = th-FAM, Q = BHQ2 L = G-clamp JJESXAP04FQ6 29FTTTTGCQGCGGACGCCCACATCCG P = phosphate, P F = th-FAM, Q = BHQ2JJESXAP04FQ5 30 FTTTTGQCGCGGACGCCCACATCCG P = phosphate, P F = th-FAM,Q = BHQ2 JJESXAP04GFQ6 31 FTTTTGCQGLGGACGCCCACATCCG P = phosphate, P F =th-FAM, Q = BHQ2 L = G-clamp JJESXAP05FQ6 32 FTTTGCGQCGGACGCCCACATCCGGP = phosphate, P F = th-FAM, Q = BHQ2 JJESXAP06FQ6 33FTTTGCGQCGGACGCCCACATCCGP P = phosphate, F = th-FAM, Q = BHQ2JJESXAP07FQ6 34 FTTTGCGQCGGACGCCCACATCCGP P = phosphate, F = th-FAM, Q =BHQ2 JJESXAP08FQ6 35 FTGTTTTQGCGCGGACGCCCACATC P = phosphate, CP F =th-FAM, Q = BHQ2 JJESXAP08FQ5 36 FTGTTTQTGCGCGGACGCCCACATC P =phosphate, CP F = th-FAM, Q = BHQ2 JJESXAP16PFQ6 37<FAM_Thr><pdU><pdU><pdU> <pdU>G<pdC><BHQ_2>G<pdC>GGA<pdC>G<pdC><pdC><pdC> A<Phos> JJESXAP17PFQ6 38<FAM_Thr><pdU><pdU><pdU> <pdU>G<pdC><BHQ_2>G<pdC>GGA<pdC>G<pdC><pdC><pdC> <Phos> JJESXAP21PFQ6 39<FAM_Thr><pdU>G<pdU><pdU> <pdU><pdU><BHQ_2>G<pdC>G<pdC>GGA<pdC>G<pdC><pdC> <pdC><Phos> JJESXAP22PFQ6 40<FAM_Thr><pdU>G<pdU><pdU> <pdU><pdU><BHQ_2>G<pdC>G<pdC>GGA<pdC>G<pdC><pdC> <Phos>

TABLE IV ESX AMPLICONS Amplicons SEQ ID Oligo Name NO: SequenceNotations esxJ amplicon 41 ATGGCCTCGCGTTTTATGACGGATC FWD Primer FP01 andregion CGCACGCGATGCGGGACATGGCGG REV Primer RP02 GCCGTTTTGAGGTGCACGCCCAGAunderlined), nucleo- CGGTGGAGGACGAGGCTCGCCGGA tides which indicateTGTGGGCGTCCGCGCAAAACATCT differences between 5 C G GG CGCGGGCTGGAGTGGCATGG copies (homologues) CCGAGGCGACCTCGCTAGACACCAof the esx gene TG A CC C AGATGAATCAGGCGTTTC (bold underlined)GCAACATCGTGAACATGCTGCACG GGGTGCGTGACGGGCTGGTTCGCG ACGCCACAACTACGAesxK amplicon 42 ATGGCCTCGCGTTTTATGACGGATC FWD Primer FP01 and regionCGCACGCGATGCGGGACATGGCGG REV Primer RP02 GCCGTTTTGAGGTGCACGCCCAGA(underlined), nucleo- CGGTGGAGGACGAGGCTCGCCGGA tides which indicateTGTGGGCGTCCGCGCAAAACAT T T differences between 5 C C GG CGCGGGCTGGAGTGGCATGG copies (homologues) CCGAGGCGACCTCGCTAGACACCAof the esx gene TG A CC C AGATGAATCAGGCGTTTC (bold underlined)GCAACATCGTGAACATGCTGCACG GGGTGCGTGACGGGCTGGTTCGCG ACGCCAACAACTACGAesxM amplicon 43 ATGGCCTCGCGTTTTATGACGGATC FWD Primer FP01 and regionCGCACGCGATGCGGGACATGGCGG REV Primer RP02 GCCGTTTTGAGGTGCACGCCAGA(underlined), nucleo- CGGTGGAGGACGAGGCTCGCCGGA tides which indicateTGTGGCGTCCGCGCAAAACAT C T differences between 5 C G GG CGCGGGCTGGAGTGGCATGG copies (homologues) CCGAGGCGACCTCGCTAGACACCAof the esx gene T G GCCC AGATGAATCAGGCGTTTC (bold underlined)GCAACATCGTGAACATGCTGCACG GGGTGCGTGACGGGCTGGTTCGCG ACGCCAACAACTACGAesxP amplicon 44 ATGGCCTCGCGTTTTATGACGGATC FWD Primer FP01 and regionCGCACGCGATGCGGGACATGGCGG REV Primer RP02 GCCGTTTTGAGGTGCACGCCCAGA(underlined), nucleo- CGGTGGAGGACGAGGCTCGCCGGA tides which indicateTGTGGGCGTCCGCGCAAAACATTT differences between 5 CCGGTGCGGGCTGGAGTGGCATGGcopies (homologues) CCGAGGCGACCTCGCTAGACACCA of the esx geneTGGCCCAGATGAATCAGGCGTTTC (bold underlined) GCAACATCGTGAACATGCTGCACGGGGTGCGTGACGGCTGGTTCGCG ACGCCAACAACTACGA esxW amplicon 45ATGGCCTCGCGTTTTATGACGGATC FWD Primer FP01 and region CGCA TGCGATGCGGGACATGGCGG REV Primer RP02 GCCGTTTTGAGGTGCACGCCCAGA(underlined), nucleo- CGGTGGAGGACGAGGCTCGCCCGGA tides which indicateTGTGGGCGTCCGCGCAAAACATTT differences between 5 C C GG TGCGGGCTGGAGTGGCATGG copies (homologues) CCGAGGCGACCTCGCTAGACACCAof the esx gene T G ACC T AGATGAATCAGGCGTTTCG (bold underlined)CAAACATCGTGAACATGCTGCACGG GGTGCGTGACGGGCTGGTTCGCGA CGCCAACAACTACGA

In one embodiment, the above described sets of esxJ primers and probesare used in order to provide for detection of MTB in a biological samplesuspected of containing MTB. The sets of primers and probes may compriseor consist of the primers and probes specific for the esxJ nucleic acidsequences, comprising or consisting of the nucleic acid sequences of SEQID NOs: 1-40. In another embodiment, the primers and probes for the esxJtarget comprise or consist of a functionally active variant of any ofthe primers and probes of SEQ ID NOs: 1-40.

A functionally active variant of any of the primers and/or probes of SEQID NOs: 1-40 may be identified by using the primers and/or probes in thedisclosed methods. A functionally active variant of a primer and/orprobe of any of the SEQ ID NOs: 1-40 pertains to a primer and/or probewhich provides a similar or higher specificity and sensitivity in thedescribed method or kit as compared to the respective sequence of SEQ IDNOs: 1-40.

The variant may, e.g., vary from the sequence of SEQ ID NOs: 1-40 by oneor more nucleotide additions, deletions or substitutions such as one ormore nucleotide additions, deletions or substitutions at the 5′ endand/or the 3′ end of the respective sequence of SEQ ID NOs: 1-40. Asdetailed above, a primer (and/or probe) may be chemically modified,i.e., a primer and/or probe may comprise a modified nucleotide or anon-nucleotide compound. A probe (or a primer) is then a modifiedoligonucleotide. “Modified nucleotides” (or “nucleotide analogs”) differfrom a natural “nucleotide” by some modification but still consist of abase or base-like compound, a pentofuranosyl sugar or a pentofuranosylsugar-like compound, a phosphate portion or phosphate-like portion, orcombinations thereof. For example, a “label” may be attached to the baseportion of a “nucleotide” whereby a “modified nucleotide” is obtained. Anatural base in a “nucleotide” may also be replaced by, e.g., a7-desazapurine whereby a “modified nucleotide” is obtained as well. Theterms “modified nucleotide” or “nucleotide analog” are usedinterchangeably in the present application. A “modified nucleoside” (or“nucleoside analog”) differs from a natural nucleoside by somemodification in the manner as outlined above for a “modified nucleotide”(or a “nucleotide analog”).

Oligonucleotides including modified oligonucleotides and oligonucleotideanalogs that amplify a nucleic acid molecule encoding the esxJ target,e.g., nucleic acids encoding alternative portions of esxJ can bedesigned using, for example, a computer program such as OLIGO (MolecularBiology Insights Inc., Cascade, Colo.). Important features whendesigning oligonucleotides to be used as amplification primers include,but are not limited to, an appropriate size amplification product tofacilitate detection (e.g., by electrophoresis), similar meltingtemperatures for the members of a pair of primers, and the length ofeach primer (i.e., the primers need to be long enough to anneal withsequence-specificity and to initiate synthesis but not so long thatfidelity's reduced during oligonucleotide synthesis). Typically,oligonucleotide primers are 8 to 50 nucleotides in length (e.g., 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, or 50 nucleotides in length).

In addition to a set of primers, the methods may use one or more probesin order to detect the presence or absence of MTB. The term “probe”refers to synthetically or biologically produced nucleic acids (DNA orRNA), which by design or selection, contain specific nucleotidesequences that allow them to hybridize under defined predeterminedstringencies specifically (i.e., preferentially) to “target nucleicacids”, in the present case to an esxJ (target) nucleic acid. A “probe”can be referred to as a “detection probe” meaning that it detects thetarget nucleic acid.

In some embodiments, the described esxJ probes can be labeled with atleast one fluorescent label. In one embodiment, the esxJ probes can belabeled with a donor fluorescent moiety, e.g., a fluorescent dye, and acorresponding acceptor moiety, e.g., a quencher. In one embodiment, theprobe comprises or consists of a fluorescent moiety and the nucleic acidsequences comprise or consist of SEQ ID NOs: 22-40.

Designing oligonucleotides to be used as probes can be performed in amanner similar to the design of primers. Embodiments may use a singleprobe or a pair of probes for detection of the amplification product.Depending on the embodiment, the probe(s) use may comprise at least onelabel and/or at least one quencher moiety. As with the primers, theprobes usually have similar melting temperatures, and the length of eachprobe must be sufficient for sequence-specific hybridization to occurbut not so long that fidelity is reduced during synthesis.Oligonucleotide probes are generally 15 to 40 (e.g., 16, 18, 20, 21, 22,23, 24, or 25) nucleotides in length.

Constructs can include vectors each containing one of esxJ primers andprobes nucleic acid molecules (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,9, and 10). Constructs can be used, for example, as control templatenucleic acid molecules. Vectors suitable for use are commerciallyavailable and/or produced by recombinant nucleic acid technology methodsroutine in the art. EsxJ nucleic acid molecules can be obtained, forexample, by chemical synthesis, direct cloning from MTB, or by PCRamplification.

Constructs suitable for use in the methods typically include, inaddition to the esxJ nucleic acid molecules (e.g., a nucleic acidmolecule that contains one or more sequences of SEQ ID NOs: 1-40),sequences encoding a selectable marker (e.g., an antibiotic resistancegene) for selecting desired constructs and/or transformants, and anorigin of replication. The choice of vector systems usually depends uponseveral factors, including, but not limited to, the choice of hostcells, replication efficiency, selectability, inducibility, and the easeof recovery.

Constructs containing esxJ nucleic acid molecules can be propagated in ahost cell. As used herein, the term host cell is meant to includeprokaryotes and eukaryotes such as yeast, plant and animal cells.Prokaryotic hosts may include E. coli, Salmonella typhimurium, Serrotiamarcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts suchas S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COScells or Chinese hamster ovary (CHO) cells, insect cells, and plantcells such as Arabidopsis thaliana and Nicotiana tabacum. A constructcan be introduced into a host cell wing any of the techniques commonlyknown to those of ordinary skill in the art. For example, calciumphosphate precipitation, electroporation, heat shock, lipofection,microinjection, and viral-mediated nucleic acid transfer are commonmethods for introducing nucleic acids into host cells. In addition,naked DNA can be delivered directly to cells (see, e.g., U.S. Pat. Nos.5,380,859 and 5,389,466).

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 discloseconventional PCR techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in some embodiments include oligonucleotidescapable of acting as points of initiation of nucleic acid synthesiswithin the described esxJ nucleic acid sequences (e.g., SEQ ID NOs:1-21). A primer can be purified from a restriction digest byconventional methods, or it can be produced synthetically. The primer ispreferably single-stranded for maximum efficiency in amplification, butthe primer can be double stranded. Double stranded primers are firstdenatured, i.e., treated to separate the strands. One method ofdenaturing double stranded nucleic acids is by heating.

If the template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5min).

If the double-stranded template nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence on the described esxJnucleic acid molecules. The temperature for annealing is usually fromabout 35° C. to about 65° C. (e.g., about 40° C. to about 60° C.; about45° C. to about 50° C.). Annealing times can be from about 10 sec toabout 1 min (e.g., about 20 sec to about 50 sec; about 30 sec to about40 sec). The reaction mixture is then adjusted to a temperature at whichthe activity of the polymerase is promoted or optimized, i.e., atemperature sufficient for extension to occur from the annealed primerto generate products complementary to the template nucleic acid. Thetemperature should be sufficient to synthesize an extension product fromeach primer that is annealed to a nucleic acid template, but should notbe so high as to denature an extension product from its complementarytemplate (e.g., the temperature for extension generally ranges fromabout 40° C. to about 80° C. (e.g., about 50° C. to about 70° C.; about60° C.). Extension times can be from about 10 sec to about 5 min (e.g.,about 30 sec to about 4 min; about 1 min to about 3 min; about 1 min 30sec to about 2 min).

PCR assays can employ esxJ nucleic acid such as RNA or DNA (cDNA). Thetemplate nucleic acid need not be purified; it may be a minor fractionof a complex mixture, such as esxJ nucleic acid contained in humancells. EsxJ nucleic acid molecules may be extracted from a biologicalsample by routine techniques such as those described in DiagnosticMolecular Microbiology: Principles and Applications (Persing et al.(eds), 1993, American Society for Microbiology, Washington D.C.).Nucleic acids can be obtained from any number of sources, such asplasmids, or natural sources including bacteria, yeast, viruses,organelles, or higher organisms such as plants or animals.

The oligonucleotide primers (e.g., SEQ ID NOs:1-24) are combined withPCR reagents under reaction conditions that induce primer extension. Forexample, chain extension reactions generally include 50 mM KCl, 10 mMTris-HCl (pH 8.3), 15 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 μgdenatured template DNA, 50 pmoles of each oligonucleotide primer, 2.5 Uof Taq polymerase, and 10% DMSO). The reactions usually contain 150 to320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

The newly synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target esxJ nucleic acid molecules. The limiting factors in thereaction are the amounts of primers, thermostable enzyme, and nucleosidetriphosphates present in the reaction. The cycling steps (i.e.,denaturation, annealing, and extension) are preferably repeated at leastonce. For use in detection, the number of cycling steps will depend,e.g., on the nature of the sample. If the sample is a complex mixture ofnucleic acids, more cycling steps will be required to amplify the targetsequence sufficient for detection. Generally, the cycling steps arerepeated at least about 20 times, hut may be repeated as many as 40, 60,or even 100 times.

Fluorescence Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on a concept that when a donorfluorescent moiety and a corresponding acceptor fluorescent moiety arepositioned within a certain distance of each other, energy transfertakes place between the two fluorescent moieties that can be visualizedor otherwise detected and/or quantitated. The donor typically transfersthe energy to the acceptor when the donor is excited by light radiationwith a suitable wavelength. The acceptor typically re-emits thetransferred energy in the form of light radiation with a differentwavelength. In certain systems, non-fluorescent energy can betransferred between donor and acceptor moieties, by way of biomoleculesthat include substantially non-fluorescent donor moieties (see, forexample, U.S. Pat. No. 7,741,467).

In one example, a oligonucleotide probe can contain a donor fluorescentmoiety and a corresponding quencher, which may or not be fluorescent,and which dissipates the transferred energy in a form other than light.When the probe is intact, energy transfer typically occurs between thedonor and acceptor moieties such that fluorescent emission from thedonor fluorescent moiety is quenched the acceptor moiety. During anextension step of a polymerase chain reaction, a probe bound to anamplification product is cleaved by the 5′ to 3′ nuclease activity of,e.g., a Taq Polymerase such that the fluorescent emission of the donorfluorescent moiety is no longer quenched. Exemplary probes for thispurpose are described in, e.g., U.S. Pat. Nos. 5,210,015, 5,994,056, and6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRApair. Commonly used quenchers are DABCYL and TAMRA. Commonly used darkquenchers include BlackHole Quenchers™ (BHQ), (Biosearch Technologies,Inc., Novato, Calif.), Iowa Black™, (Integrated DNA Tech., Inc.,Coralville, Iowa), BlackBerry™ Quencher 650 (BBQ-650), (Berry & Assoc.,Dexter, Mich.).

In another example, two oligonucleotide probes, each containing afluorescent moiety, can hybridize to an amplification product atparticular positions determined by the complementarity of theoligonucleotide probes to the esxJ target nucleic acid sequence. Uponhybridization of the oligonucleotide probes to the amplification productnucleic acid at the appropriate positions, a FRET signal is generated.Hybridization temperatures can range from about 35° C. to about 65° C.for about 10 sec to about 1 min.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system, or afluorimeter. Excitation to initiate energy transfer, or to allow directdetection of a fluorophore, can be carried out with an argon ion laser,a high intensity mercury (Hg) arc lamp, a fiber optic light source, orother high intensity light source appropriately filtered for excitationin the desired range.

As used herein with respect to donor and corresponding acceptor moieties“corresponding” refers to an acceptor fluorescent moiety or a darkquencher having an absorbance spectrum that overlaps the emissionspectrum of the donor fluorescent moiety. The wavelength maximum of theemission spectrum of the acceptor fluorescent moiety should be at least100 nm greater than the wavelength maximum of the excitation spectrum ofthe donor fluorescent moiety. Accordingly, efficient non-radiativeenergy transfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Forster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate, or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm can be the distance in Angstroms (Å) from the nucleotide baseto the fluorescent moiety. In general, a linker arm is from about 10 Åto about 25 Å. The linker arm may be of the kind described in WO84/03285, WO 84/03285 also discloses methods for attaching linker armsto a particular nucleotide base, and also for attaching fluorescentmoieties to a linker arm.

An acceptor fluorescent moiety, such as an LC Red 640, can be combinedwith an oligonucleotide which contains an amino linker (e.g., C6-aminophosphoramidites available from ABI (Foster City, Calif.) or GlenResearch (Sterling, Va.)) to produce, for example, LC Red 640 labeledoligonucleotide. Frequently used linkers to couple a donor fluorescentmoiety such as fluorescein to an oligonucleotide include thiourealinkers (FITC-derived, for example, fluorescein-CPG's from Glen Researchor ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPGs that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of MTB

The present disclosure provides methods for detecting the presence orabsence of MTB in a biological or non-biological sample. Methodsprovided avoid problems of sample contamination, false negatives, andfalse positives. The methods include performing at least one cyclingstep that includes amplifying a portion of esxJ target nucleic acidmolecules from a sample using one or more pairs of esxJ primers, and aFRET detecting step. Multiple cycling steps are performed, preferably ina thermocycler. Methods can be performed using the esxJ primers andprobes to detect the presence of MTB, and the detection of esxJindicates the presence of MTB in the sample.

As described herein, amplification products can be detected usinglabeled hybridization probes that take advantage of FRET technology. OneFRET format utilizes TaqMan® technology to detect the presence orabsence of an amplification product, and hence, the presence or absenceof MTB. TaqMan® technology utilizes one single-stranded hybridizationprobe labeled with, e.g., one fluorescent dye and one quencher, whichmay or may not be fluorescent. When a first fluorescent moiety isexcited with light of a suitable wavelength, the absorbed energy istransferred to a second fluorescent moiety or a dark quencher accordingto the principles of FRET. The second moiety is generally a quenchermolecule. During the annealing step of the PCR reaction, the labeledhybridization probe binds to the target DNA (i.e., the amplificationproduct) and is degraded by the 5′ to 3′ nuclease activity of, e.g., theTaq Polymerase during the subsequent elongation phase. As a result, thefluorescent moiety and the quencher moiety become spatially separatedfrom one another. As a consequence, upon excitation of the firstfluorescent moiety in the absence of the quencher, the fluorescenceemission from the first fluorescent moiety can be detected. By way ofexample, an ABI PRISM® 7700 Sequence Detection System (AppliedBiosystems) uses TaqMan® technology, and is suitable for performing themethods described herein for detecting the presence or absence of MTB inthe sample.

Molecular beacons in conjunction with FRET can also be used to detectthe presence of an amplification product using the real time PCRmethods. Molecular beacon technology uses a hybridization probe labeledwith a first fluorescent moiety and a second fluorescent moiety. Thesecond fluorescent moiety is generally a quencher, and the fluorescentlabels are typically located at each end of the probe. Molecular beacontechnology uses a probe oligonucleotide having sequences that permitsecondary structure formation (e.g., a hairpin). As a result ofsecondary structure formation within the probe, both fluorescentmoieties are in spatial proximity when the probe is in solution. Afterhybridization to the target nucleic acids (i.e., amplificationproducts), the secondary structure of the probe is disrupted and thefluorescent moieties become separated from one another such that afterexcitation with light of a suitable wavelength, the emission of thefirst fluorescent moiety can be detected.

Another common format of FRET technology utilizes two hybridizationprobes. Each probe can be labeled with a different fluorescent moietyand are generally designed to hybridize in close proximity to each otherin a target DNA molecule (e.g., an amplification product). A donorfluorescent moiety, for example, fluorescein, is excited at 470 nm bythe light source of the LightCycler® Instrument. During FRET, thefluorescein transfers its energy to an acceptor fluorescent moiety suchas LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red705). The acceptor fluorescent moiety then emits light of a longerwavelength, which is detected by the optical detection system of theLightCycler® instrument. Efficient FRET can only take place when thefluorescent moieties are in direct local proximity and when the emissionspectrum of the donor fluorescent moiety overlaps with the absorptionspectrum of the acceptor fluorescent moiety. The intensity of theemitted signal can be correlated with the number of original target DNAmolecules (e.g., the number of MTB genomes). If amplification of esxJtarget nucleic acid occurs and an amplification product is produced, thestep of hybridizing results in a detectable signal based upon FRETbetween the members of the pair of probes.

Generally, the presence of FRET indicates the presence of MTB in thesample, and the absence of FRET indicates the absence of MTB in thesample. Inadequate specimen collection, transportation delays,inappropriate transportation conditions, or use of certain collectionswabs (calcium alginate or aluminum shaft) are all conditions that canaffect the success and/or accuracy of a test result, however. Using themethods disclosed herein, detection of FRET within, e.g., 45 cyclingsteps is indicative of a MTB infection.

Representative biological samples that can be used in practicing themethods include, but are not limited to respiratory specimens, fecalspecimens, blood specimens, dermal swabs, nasal swabs, wound swabs,blood cultures, skin, and soft tissue infections. Collection and storagemethods of biological samples are known to those of skill in the art.Biological samples can be processed (e.g., by nucleic acid extractionmethods and/or kits known in the art) to release MTB nucleic acid or insome cases, the biological sample can be contacted directly with the PCRreaction components and the appropriate oligonucleotides.

Melting curve analysis is an additional step that can be included in acycling profile. Melting curve analysis is based on the fact that DNAmelts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Similarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the esxJ probes from the esxJ amplificationproducts can confirm the presence or absence of MTB in the sample.

Within each thermocycler run, control samples can be cycled as well.Positive control samples can amplify target nucleic acid controltemplate (other than described amplification products of target genes)using, for example, control primers and control probes. Positive controlsamples can also amplify, for example, a plasmid construct containingthe target nucleic acid molecules. Such a plasmid control can beamplified internally (e.g., within the sample) or in a separate samplerun side by side with the patients' samples using the same primers andprobe as used for detection of the intended target. Such controls areindicators of the success or failure of the amplification,hybridization, and/or FRET reaction. Each thermocycler run can alsoinclude a negative control that, for example, lacks target template DNA.Negative control can measure contamination. This ensures that the systemand reagents would not give rise to a false positive signal. Therefore,control reactions can readily determine, for example, the ability ofprinters to anneal with sequence-specificity and to initiate elongation,as well as the ability of probes to hybridize with sequence-specificityand for FRET to occur.

In an embodiment, the methods include steps to avoid contamination. Forexample, an enzymatic method utilizing uracil-DNA glycosylase isdescribed in U.S. Pat. Nos. 5,035,996, 5,683,896 and 5,945,313 to reduceor eliminate contamination between one thermocycler run and the next.

Conventional PCR methods in conjunction with FRET technology can be usedto practice the methods. In one embodiment, a LightCycler® instrument isused. The following patent applications describe real-time PCR as usedin the LightCycler® technology: WO 97/46707, WO 97/46714, and WO97/46712.

The LightCycler® can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 milliseconds (msec). After each cycling step, a quantitativedisplay of fluorescence vs. cycle number can be continually updated forall samples. The data generated can be stored for further analysis.

As an alternative to FRET, an amplification product can be detectedusing a double-stranded DNA binding dye such as a fluorescent DNAbinding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). Uponinteraction with the double-stranded nucleic acid, such fluorescent DNAbinding dyes emit a fluorescence signal after excitation with light at asuitable wavelength. A double-stranded DNA binding dye such as a nucleicacid intercalating dye also can be used. When double-stranded DNAbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

It is understood that the embodiments of the present disclosure are notlimited by the configuration of one or more commercially availableinstruments.

Articles of Manufacture/Kits

Embodiments of the present disclosure further provide for articles ofmanufacture or kits to detect MTB. An article of manufacture can includeprimers and probes used to detect the esxJ gene target, together withsuitable packaging materials. Representative primers and probes fordetection of MTB are capable of hybridizing to esxJ target nucleic acidmolecules. In addition, the kits may also include suitably packagedreagents and materials needed for DNA immobilization, hybridization, anddetection, such solid supports, buffers, enzymes, and DNA standards.Methods of designing primers and probes are disclosed herein, andrepresentative examples of primers and probes that amplify and hybridizeto esxJ target nucleic add molecules are provided.

Articles of manufacture can also include one or more fluorescentmoieties for labeling the probes or, alternatively, the probes suppliedwith the kit can be labeled. For example, an article of manufacture mayinclude a donor and/or an acceptor fluorescent moiety for labeling theesxJ probes. Examples of suitable FRET donor fluorescent moieties andcorresponding acceptor fluorescent moieties are provided above.

Articles of manufacture can also contain a package insert or packagelabel having instructions thereon for using the esxJ primers and probesto detect MTB in a sample. Articles of manufacture may additionallyinclude reagents for carrying out the methods disclosed herein (e.g.,buffers, polymerase enzymes, co-factors, or agents to preventcontamination). Such reagents may be specific for one of thecommercially available instruments described herein.

Embodiments of the present disclosure will be further described in thefollowing examples, which do not limit the scope of the inventiondescribed in the claims.

EXAMPLES

The following examples and figures are provided to aid the understandingof the subject matter, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Example I

Specificity for oligos targeting the esxJ gene was established by blastanalysis of 28 MTB whole genomes available publicly, as well as 26 wholegenomes of non-tuberculous Mycobacteria. The targeted gene region waspresent with 5 distinct copies in all 28 MTB whole genomes as well aspresent with 3-5 distinct copies in other member of the MTB complex (M.bovis, M. africanum, and M. canettii), and only found in otherMycobacterial species with very poor homology. Exclusivity was confirmedby testing genomic DNA extracted from numerous non-tuberculousMycobacterial species.

Referring to FIG. 1, MTB esx forward primers were screened indicatingsimilar elbow values and higher fluorescence from FP07 and FP09 comparedto FP01. Referring to FIG. 2, MTB esx reverse primers were screenedindicating earlier elbow values and higher fluorescence from all threetop esx primer candidates compared to 16S optimized oligos which targeta single copy genomic location. Referring to FIG. 3, MTB esx probescreen shows fluorescence and elbow values from top 4 candidates withMTB target. All probes yielded greater than 12 units of fluorescence.Referring to FIG. 4, MTB esx probe screen showing fluorescence from top4 candidates with non-MTB target (M. gastri). Due to observed crossreactivity with M. gastri, highest yielding probes were eliminated fromcandidacy. Referring to FIG. 5, exclusivity demonstration of top esxoligonucleotides with dilution series of MTB and 1e6c/PCR each ofnon-MTB species (M. gastri, M. szulgai, and M. kansasii).

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed:
 1. A method of detecting Mycobacterium tuberculosis(MTB) and other members of the MTB-complex in a sample, the methodcomprising: performing an amplifying step comprising contacting thesample with a set of esxJ primers to produce an amplification product ifa esxJ nucleic acid is present in the sample; performing a hybridizingstep comprising contacting the amplification product with one or moredetectable esxJ probes; and detecting the presence or absence of theamplification product, wherein the presence of the amplification productis indicative of the presence of MTB in the sample and wherein theabsence of the amplification product is indicative of the absence of MTBin the sample; wherein the set of esxJ primers comprise a first primerconsisting of SEQ ID NO: 1, or the complement thereof, and optionallyhaving at least one modified nucleotide, or SEQ ID NO: 7, or thecomplement thereof, and optionally having at least one modifiednucleotide, or SEQ ID NO: 8, or the complement thereof, and optionallyhaving at least one modified nucleotide; and a second primer consistingof SEQ ID NO: 15, or the complement thereof, and optionally having atleast one modified nucleotide, or SEQ ID NO: 16, or the complement,thereof and optionally having at least one modified nucleotide, or SEQID NO: 17, or the complement thereof, and optionally having at least onemodified nucleotide; and wherein one of the one or more detectable esxJprobes consists of SEQ ID NO: 30, or the complement thereof, andoptionally having at least one modified nucleotide, at least one label,and at least one quencher moiety; and wherein the set of esxJ primersand the one or more detectable esxJ probes used in combination do notamplify and/or detect non-MTB species in the sample.
 2. The method ofclaim 1, wherein: the hybridizing step comprises contacting theamplification product with the detectable esxJ probe that is labeledwith a donor fluorescent moiety and a corresponding acceptor moiety; andthe detecting step comprises detecting the presence or absence offluorescence resonance energy transfer (FRET) between the donorfluorescent moiety and the acceptor moiety of the probe, wherein thepresence or absence of fluorescence is indicative of the presence orabsence of MTB in the sample.
 3. The method of claim 2, wherein saidamplification step employs a polymerase enzyme having 5′ to 3′ nucleaseactivity.
 4. The method of claim 2, wherein the acceptor moiety is aquencher.
 5. The method of claim 1, wherein any one of the set of esxJprimers and the one or more detectable esxJ probes comprise at least onemodified nucleotide.
 6. The method of claim 5, wherein the at least onemodified nucleotide is selected from t-butylbenzyldeoxyadenine ort-butylbenzyldeoxycytosine.
 7. The method of claim 1 wherein the firstprimer consists of SEQ ID NO: 1, and contains t-butylbenzyldeoxyadenineon its 3′ terminal nucleotide.
 8. The method of claim 1 wherein thefirst primer consists of SEQ ID NO: 7 and containst-butylbenzyldeoxycytosine on its 3′ terminal nucleotide.
 9. The methodof claim 1 wherein the first primer consists of SEQ ID NO: 8 andcontains t-butylbenzyldeoxycytosine on its 3′ terminal nucleotide. 10.The method of claim 1 wherein the second primer consists of SEQ ID NO:15 and contains t-butylbenzyldeoxycytosine on its 3′ terminalnucleotide.
 11. The method of claim 1 wherein the second primer consistsof SEQ ID NO: 16 and contains t-butylbenzyldeoxycytosine on its 3′terminal nucleotide.
 12. The method of claim 1 wherein the second primerconsists of SEQ ID NO: 17 and contains t-butylbenzyldeoxycytosine on its3′ terminal nucleotide.